Association of FHOD2 with common type 2 diabetes mellitus

Abstract

FHOD2 has been identified as a type 2 diabetes susceptibility gene. Methods for diagnosing and treating type 2 diabetes and methods for identifying compounds for use in the diagnosis and treatment of diabetes are disclosed. Improved diagnostic methods for early detection of a risk for developing type 2 diabetes mellitus in humans, and screening assays for therapeutic agents useful in the treatment of type 2 diabetes mellitus, by analyzing the FHOD2 gene or gene products from FHOD2, including variants forms of FHOD2, are disclosed. Indicators of diabetes include variant forms of the FHOD2 protein, variant forms of FHOD2 pre-mRNA or mRNA or variant forms of the genomic DNA of the FHOD2 gene or DNA surrounding FHOD2.

Description

RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Application No. 60/495,624 filed Aug. 15, 2003, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention is related to a genomic region that is linked to type 2 diabetes. Methods of identifying patients at risk of developing diabetes or impaired fasting glucose (IFG), or impaired glucose tolerance (IGT) (together termed “impaired glucose homeostasis” [IH]) are disclosed.

BACKGROUND OF THE INVENTION

Type 2 diabetes is a disease of unknown molecular basis but thought to result from defects in insulin action and insulin secretion. Twin studies indicate that there is a genetic component to the disease but the genetic defects responsible for the majority of cases have not yet been identified. For some relatively uncommon variants of the disease, causative mutations have been identified. For example, mutations have been found in the insulin and insulin-receptor genes, in glucokinase and in the HNF-4α and HNF-1′α transcription factors. The disease is thought to be oligogenic, with two or more genes contributing to the phenotype.

SUMMARY OF THE INVENTION

A human gene that is linked to type 2 diabetes melittus is disclosed. The gene, FHOD2, also known as FMNL2, may be used as a target to identify drugs to treat type 2 diabetes. The expression of the gene may be used as a method of diagnosing diabetes or of identifying individuals who are at risk for developing diabetes or impaired fasting glucose (IFG), or impaired glucose tolerance (IGT) (together termed “impaired glucose homeostasis” [IH]). Polymorphisms in the FHOD2 gene or in the region surrounding FHOD2 may be used to identify individuals who are at risk for diabetes or IH. A haplotype that is associated with diabetes and includes at least a portion of the FHOD2 gene may be identified and used as an indicator of individuals who are at risk for diabetes.

In some embodiments any 25 contiguous bases of SEQ ID NO 2 or any 25 contiguous bases of the complement to SEQ ID NO 2 is disclosed. In some embodiments any 25 contiguous bases of accession number NT_—052905 or its complement are disclosed. In some embodiments probes that are complementary to the FHOD2 gene, mRNA or pre-mRNA are used to detect the FHOD2 gene or gene products. Also incorporated by reference is accession number NT_—005403 which includes the sequence of a chromosome 2 genomic contig that contains FHOD2/FMNL2.

Genes in the FHOD2 gene pathway may be identified as indicators of risk for or as diagnostic of type 2 diabetes. The FHOD2 gene may be analyzed at the RNA level as pre mRNA or mRNA. The expression level of the gene may be analyzed. FHOD2 protein levels may be analyzed. An antibody to FHOD2 may be used to detect the FOD 2 protein. Antibodies to variant forms of FHOD2 may be used to detect variant forms of the FHOD2 protein. Variant forms of FHOD2 that are associated with diabetes may be detected to identify individuals at risk for diabetes or IH. Variant forms of the FHOD2 DNA or RNA may be detected to identify individuals at risk for diabetes or IH. Proteins that interact with FHOD2 or variant forms of FHOD2 may be identified. Variant forms of the FHOD2 that associate differentially with FHOD2 associated factors may be identified.

DETAILED DESCRIPTION

a) General

The present invention has many preferred embodiments and relies on many patents, applications and other references for details known to those of the art. Therefore, when a patent, application, or other reference is cited or repeated below, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited.

As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

An individual is not limited to a human being but may also be other organisms including but not limited to mammals, plants, bacteria, or cells derived from any of the above.

Throughout this disclosure, various aspects of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3^rd Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5^th Ed., W. H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.

The present invention can employ solid substrates, including arrays in some preferred embodiments. Methods and techniques applicable to polymer (including protein) array synthesis have been described in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730 (International Publication Number WO 99/36760) and PCT/US01/04285 (International Publication Number WO 01/58593), which are all incorporated herein by reference in their entirety for all purposes.

Patents that describe synthesis techniques in specific embodiments include U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and 5,959,098. Nucleic acid arrays are described in many of the above patents, but the same techniques are applied to polypeptide arrays.

Nucleic acid arrays that are useful in the present invention include those that are commercially available from Affymetrix (Santa Clara, Calif.) under the brand name GeneChip®. Example arrays are shown on the website at affymetrix.com.

The present invention also contemplates many uses for polymers attached to solid substrates. These uses include gene expression monitoring, profiling, library screening, genotyping and diagnostics. Gene expression monitoring and profiling methods can be shown in U.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248 and 6,309,822. Genotyping and uses thereof are shown in U.S. Ser. Nos. 60/319,253, 10/013,598 (U.S. Patent Application Publication 20030036069), and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses are embodied in U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.

The present invention also contemplates sample preparation methods in certain preferred embodiments. Prior to or concurrent with genotyping, the genomic sample may be amplified by a variety of mechanisms, some of which may employ PCR. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188,and 5,333,675, and each of which is incorporated herein by reference in their entireties for all purposes. The sample may be amplified on the array. See, for example, U.S. Pat. No. 6,300,070 and U.S. Ser. No. 09/513,300, which are incorporated herein by reference.

Other suitable amplification methods include the ligase chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315), self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selective amplification of target polynucleotide sequences (U.S. Pat. No 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. No. 5,413,909, 5,861,245) and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporated herein by reference). Other amplification methods that may be used are described in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317, each of which is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing the complexity of a nucleic sample are described in Dong et al., Genome Research 11, 1418 (2001), in U.S. Pat. No. 6,361,947, 6,391,592 and U.S. Ser. Nos. 09/916,135, 09/920,491 (U.S. Patent Application Publication 20030096235), Ser. No. 09/910,292 (U.S. Patent Application Publication 20030082543), and Ser. No. 10/013,598.

Methods for conducting polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al. Molecular Cloning: A Laboratory Manual (2^nd Ed. Cold Spring Harbor, N.Y, 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying out repeated and controlled hybridization reactions have been described in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which are incorporated herein by reference

The present invention also contemplates signal detection of hybridization between ligands in certain preferred embodiments. See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. No. 60/364,731 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensity data are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. No. 60/364,731 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.

The practice of the present invention may also employ conventional biology methods, software and systems. Computer software products of the invention typically include computer readable medium having computer-executable instructions for performing the logic steps of the method of the invention. Suitable computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The computer executable instructions may be written in a suitable computer language or combination of several languages. Basic computational biology methods are described in, e.g. Setubal and Meidanis et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2^nd ed., 2001). See U.S. Pat. No. 6,420,108.

The present invention may also make use of various computer program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See, U.S. Pat. Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments that include methods for providing genetic information over networks such as the Internet as shown in U.S. Ser. Nos. 10/063,559 (United States Publication No. US20020183936), 60/349,546, 60/376,003, 60/394,574 and 60/403,381.

b) Definitions

The term “admixture” refers to the phenomenon of gene flow between populations resulting from migration. Admixture can create linkage disequilibrium (LD).

The term “allele’ as used herein is any one of a number of alternative forms a given locus (position) on a chromosome. An allele may be used to indicate one form of a polymorphism, for example, a biallelic SNP may have possible alleles A and B. An allele may also be used to indicate a particular combination of alleles of two or more SNPs in a given gene or chromosomal segment. The frequency of an allele in a population is the number of times that specific allele appears divided by the total number of alleles of that locus.

The term “array” as used herein refers to an intentionally created collection of molecules which can be prepared either synthetically or biosynthetically. The molecules in the array can be identical or different from each other. The array can assume a variety of formats, for example, libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports.

The term “biomonomer” as used herein refers to a single unit of biopolymer, which can be linked with the same or other biomonomers to form a biopolymer (for example, a single amino acid or nucleotide with two linking groups one or both of which may have removable protecting groups) or a single unit which is not part of a biopolymer. Thus, for example, a nucleotide is a biomonomer within an oligonucleotide biopolymer, and an amino acid is a biomonomer within a protein or peptide biopolymer; avidin, biotin, antibodies, antibody fragments, etc., for example, are also biomonomers.

The term “biopolymer” or sometimes refer by “biological polymer” as used herein is intended to mean repeating units of biological or chemical moieties. Representative biopolymers include, but are not limited to, nucleic acids, oligonucleotides, amino acids, proteins, peptides, hormones, oligosaccharides, lipids, glycolipids, lipopolysaccharides, phospholipids, synthetic analogues of the foregoing, including, but not limited to, inverted nucleotides, peptide nucleic acids, Meta-DNA, and combinations of the above.

The term “biopolymer synthesis” as used herein is intended to encompass the synthetic production, both organic and inorganic, of a biopolymer. Related to a bioploymer is a “biomonomer”.

The term “combinatorial synthesis strategy” as used herein refers to a combinatorial synthesis strategy is an ordered strategy for parallel synthesis of diverse polymer sequences by sequential addition of reagents which may be represented by a reactant matrix and a switch matrix, the product of which is a product matrix. A reactant matrix is a l column by m row matrix of the building blocks to be added. The switch matrix is all or a subset of the binary numbers, preferably ordered, between l and m arranged in columns. A “binary strategy” is one in which at least two successive steps illuminate a portion, often half, of a region of interest on the substrate. In a binary synthesis strategy, all possible compounds which can be formed from an ordered set of reactants are formed. In most preferred embodiments, binary synthesis refers to a synthesis strategy which also factors a previous addition step. For example, a strategy in which a switch matrix for a masking strategy halves regions that were previously illuminated, illuminating about half of the previously illuminated region and protecting the remaining half (while also protecting about half of previously protected regions and illuminating about half of previously protected regions). It will be recognized that binary rounds may be interspersed with non-binary rounds and that only a portion of a substrate may be subjected to a binary scheme. A combinatorial “masking” strategy is a synthesis which uses light or other spatially selective deprotecting or activating agents to remove protecting groups from materials for addition of other materials such as amino acids.

The term “complementary” as used herein refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. Alternatively, complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984), incorporated herein by reference.

The term “effective amount” as used herein refers to an amount sufficient to induce a desired result.

The term “genome” as used herein is all the genetic material in the chromosomes of an organism. DNA derived from the genetic material in the chromosomes of a particular organism is genomic DNA. A genomic library is a collection of clones made from a set of randomly generated overlapping DNA fragments representing the entire genome of an organism.

The term “genotype” as used herein refers to the genetic information an individual carries at one or more positions in the genome. A genotype may refer to the information present at a single polymorphism, for example, a single SNP. For example, if a SNP is biallelic and can be either an A or a C then if an individual is homozygous for A at that position the genotype of the SNP is homozygous A or AA. Genotype may also refer to the information present at a plurality of polymorphic positions.

The term “Hardy-Weinberg equilibrium” (HWE) as used herein refers to the principle that an allele that when homozygous leads to a disorder that prevents the individual from reproducing does not disappear from the population but remains present in a population in the undetectable heterozygous state at a constant allele frequency.

The term “hybridization” as used herein refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide; triple-stranded hybridization is also theoretically possible. The resulting (usually) double-stranded polynucleotide is a “hybrid.” The proportion of the population of polynucleotides that forms stable hybrids is referred to herein as the “degree of hybridization.” Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than about 1 M and a temperature of at least 25° C. For example, conditions of 5× SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C. are suitable for allele-specific probe hybridizations or conditions of 100 mM MES, 1 M [Na⁺], 20 mM EDTA, 0.01% Tween-20 and a temperature of 30-50° C., preferably at about 45-50° C. Hybridizations may be performed in the presence of agents such as herring sperm DNA at about 0.1 mg/ml, acetylated BSA at about 0.5 mg/ml. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. Hybridization conditions suitable for microarrays are described in the Gene Expression Technical Manual, 2004 and the GeneChip Mapping Assay Manual, 2004.

The term “hybridization probes” as used herein are oligonucleotides capable of binding in a base-specific manner to a complementary strand of nucleic acid. Such probes include peptide nucleic acids, as described in Nielsen et al., Science 254, 1497-1500 (1991), LNAs, as described in Koshkin et al. Tetrahedron 54:3607-3630, 1998, and U.S. Pat. No. 6,268,490 and other nucleic acid analogs and nucleic acid mimetics.

The term “hybridizing specifically to” as used herein refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (for example, total cellular) DNA or RNA.

The term “isolated nucleic acid” as used herein mean an object species invention that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). Preferably, an isolated nucleic acid comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods).

The term “ligand” as used herein refers to a molecule that is recognized by a particular receptor. The agent bound by or reacting with a receptor is called a “ligand,” a term which is definitionally meaningful only in terms of its counterpart receptor. The term “ligand” does not imply any particular molecular size or other structural or compositional feature other than that the substance in question is capable of binding or otherwise interacting with the receptor. Also, a ligand may serve either as the natural ligand to which the receptor binds, or as a functional analogue that may act as an agonist or antagonist. Examples of ligands that can be investigated by this invention include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (for example, opiates, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, substrate analogs, transition state analogs, cofactors, drugs, proteins, and antibodies.

The term “linkage analysis” as used herein refers to a method of genetic analysis in which data are collected from affected families, and regions of the genome are identified that co-segregated with the disease in many independent families or over many generations of an extended pedigree. A disease locus may be identified because it lies in a region of the genome that is shared by all affected members of a pedigree.

The term “linkage disequilibrium” or sometimes referred to as “allelic association” as used herein refers to the preferential association of a particular allele or genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population. For example, if locus X has alleles A and B, which occur equally frequently, and linked locus Y has alleles C and D, which occur equally frequently, one would expect the combination AC to occur with a frequency of 0.25. If AC occurs more frequently, then alleles A and C are in linkage disequilibrium. Linkage disequilibrium may result from natural selection of certain combination of alleles or because an allele has been introduced into a population too recently to have reached equilibrium with linked alleles. The genetic interval around a disease locus may be narrowed by detecting disequilibrium between nearby markers and the disease locus. For additional information on linkage disequilibrium see Ardlie et al., Nat. Rev. Gen. 3:299-309, 2002.

The term “lod score” or “LOD” is the log of the odds ratio of the probability of the data occurring under the specific hypothesis relative to the null hypothesis. LOD=log [probability assuming linkage/probability assuming no linkage].

The term “mixed population” or sometimes refer by “complex population” as used herein refers to any sample containing both desired and undesired nucleic acids. As a non-limiting example, a complex population of nucleic acids may be total genomic DNA, total genomic RNA or a combination thereof. Moreover, a complex population of nucleic acids may have been enriched for a given population but include other undesirable populations. For example, a complex population of nucleic acids may be a sample which has been enriched for desired messenger RNA (mRNA) sequences but still includes some undesired ribosomal RNA sequences (rRNA).

The term “monomer” as used herein refers to any member of the set of molecules that can be joined together to form an oligomer or polymer. The set of monomers useful in the present invention includes, but is not restricted to, for the example of (poly)peptide synthesis, the set of L-amino acids, D-amino acids, or synthetic amino acids. As used herein, “monomer” refers to any member of a basis set for synthesis of an oligomer. For example, dimers of L-amino acids form a basis set of 400 “monomers” for synthesis of polypeptides. Different basis sets of monomers may be used at successive steps in the synthesis of a polymer. The term “monomer” also refers to a chemical subunit that can be combined with a different chemical subunit to form a compound larger than either subunit alone.

The term “mRNA” or sometimes refer by “mRNA transcripts” as used herein, include, but not limited to pre-mRNA transcript(s), transcript processing intermediates, mature mRNA(s) ready for translation and transcripts of the gene or genes, or nucleic acids derived from the mRNA transcript(s). Transcript processing may include splicing, editing and degradation. As used herein, a nucleic acid derived from an mRNA transcript refers to a nucleic acid for whose synthesis the mRNA transcript or a subsequence thereof has ultimately served as a template. Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc., are all derived from the mRNA transcript and detection of such derived products is indicative of the presence and/or abundance of the original transcript in a sample. Thus, mRNA derived samples include, but are not limited to, mRNA transcripts of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNA transcribed from amplified DNA, and the like.

The term “nucleic acid library” or sometimes refer by “array” as used herein refers to an intentionally created collection of nucleic acids which can be prepared either synthetically or biosynthetically and screened for biological activity in a variety of different formats (for example, libraries of soluble molecules; and libraries of oligos tethered to resin beads, silica chips, or other solid supports). Additionally, the term “array” is meant to include those libraries of nucleic acids which can be prepared by spotting nucleic acids of essentially any length (for example, from 1 to about 1000 nucleotide monomers in length) onto a substrate. The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs), that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. Thus the terms nucleoside, nucleotide, deoxynucleoside and deoxynucleotide generally include analogs such as those described herein. These analogs are those molecules having some structural features in common with a naturally occurring nucleoside or nucleotide such that when incorporated into a nucleic acid or oligonucleoside sequence, they allow hybridization with a naturally occurring nucleic acid sequence in solution. Typically, these analogs are derived from naturally occurring nucleosides and nucleotides by replacing and/or modifying the base, the ribose or the phosphodiester moiety. The changes can be tailor made to stabilize or destabilize hybrid formation or enhance the specificity of hybridization with a complementary nucleic acid sequence as desired.

The term “nucleic acids” as used herein may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. See Albert L. Lehninger, PRINCIPLES OF BIOCHEMISTRY, at 793-800 (Worth Pub. 1982). Indeed, the present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glucosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogeneous in composition, and may be isolated from naturally-occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

The term “oligonucleotide” or sometimes refer by “polynucleotide” as used herein refers to a nucleic acid ranging from at least 2, preferable at least 8, and more preferably at least 20 nucleotides in length or a compound that specifically hybridizes to a polynucleotide. Polynucleotides of the present invention include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) which may be isolated from natural sources, recombinantly produced or artificially synthesized and mimetics thereof. A further example of a polynucleotide of the present invention may be peptide nucleic acid (PNA). The invention also encompasses situations in which there is a nontraditional base pairing such as Hoogsteen base pairing which has been identified in certain tRNA molecules and postulated to exist in a triple helix. “Polynucleotide” and “oligonucleotide” are used interchangeably in this application.

The term “polymorphism” as used herein refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker or site is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population. A polymorphism may comprise one or more base changes, an insertion, a repeat, or a deletion. A polymorphic locus may be as small as one base pair. Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu. The first identified allelic form is arbitrarily designated as the reference form and other allelic forms are designated as alternative or variant alleles. The allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous for allelic forms. A diallelic polymorphism has two forms. A triallelic polymorphism has three forms. Single nucleotide polymorphisms (SNPs) are included in polymorphisms.

The term “primer” as used herein refers to a single-stranded oligonucleotide capable of acting as a point of initiation for template-directed DNA synthesis under suitable conditions for example, buffer and temperature, in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, for example, DNA or RNA polymerase or reverse transcriptase. The length of the primer, in any given case, depends on, for example, the intended use of the primer, and generally ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with such template. The primer site is the area of the template to which a primer hybridizes. The primer pair is a set of primers including a 5′ upstream primer that hybridizes with the 5′ end of the sequence to be amplified and a 3′ downstream primer that hybridizes with the complement of the 3′ end of the sequence to be amplified.

The term “probe” as used herein refers to a surface-immobilized molecule that can be recognized by a particular target. See U.S. Pat. No. 6,582,908 for an example of arrays having all possible combinations of probes with 10, 12, and more bases. Examples of probes that can be investigated by this invention include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (for example, opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.

The term “receptor” as used herein refers to a molecule that has an affinity for a given ligand. Receptors may be naturally-occurring or manmade molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Receptors may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of receptors which can be employed by this invention include, but are not restricted to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. Receptors are sometimes referred to in the art as anti-ligands. As the term receptors is used herein, no difference in meaning is intended. A “Ligand Receptor Pair” is formed when two macromolecules have combined through molecular recognition to form a complex. Other examples of receptors which can be investigated by this invention include but are not restricted to those molecules shown in U.S. Pat. No. 5,143,854, which is hereby incorporated by reference in its entirety.

The term “solid support”, “support”, and “substrate” as used herein are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. See U.S. Pat. No. 5,744,305 for exemplary substrates.

The term “target” as used herein refers to a molecule that has an affinity for a given probe. Targets may be naturally-occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Targets may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of targets which can be employed by this invention include, but are not restricted to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, oligonucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. Targets are sometimes referred to in the art as anti-probes. As the term targets is used herein, no difference in meaning is intended. A “Probe Target Pair” is formed when two macromolecules have combined through molecular recognition to form a complex.

FMNL2/FHOD2 is Associated with Common Type 2 Diabetes Mellitus

Diabetes mellitus is a high prevalence illness characterized by high blood glucose levels. The chronic hyperglycemia (high glucose level) of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels.

The vast majority of cases of diabetes fall into two broad etiopathogenetic categories. The first category, type 1 or insulin-dependent diabetes mellitus (IDDM), results from an absolute deficiency of insulin due to autoimmunological destruction of the insulin-producing pancreatic β-cells. Another category, type 2 or non-insulin-dependent diabetes mellitus (NIDDM), which accounts for ˜90% of all diabetes cases, is caused by a combination of resistance of insulin action and an inadequate compensatory insulin secretory response.

The genetic defects responsible for the vast majority of cases of type 2 diabetes have not been identified except for a rare, monogenic form of type 2 diabetes-maturity-onset diabetes of the young (MODY). MODY is characterized by non-ketotic diabetes mellitus, an autosomal dominant mode of inheritance, onset typically before 25 years of age and primary defects in pancreatic β-cell dysfunction. Genetically, it results from mutations in any one of the genes encoding for the glycolytic enzyme glucokinase on chromosome 7 and five transcription factors mapped to chromosome 12 and 20. MODY genes are thought to play, at most, a minor role in common type 2 diabetes.

Linkage analysis for families with multiple affected members can identify the genes that predispose the individual to disease. The identified gene may then be used to identify the biochemical or regulatory pathways involved in pathogenesis. Polymorphisms in the gene may be identified to determine which are associated with the disease phenotype and which may confer protection against the disease phenotype. Known polymorphisms in and near the gene may be genotyped to narrow the region of interest and to identify polymorphisms linked to the disease phenotype. The region may be resequenced in affected individuals to identify novel polymorphisms associated with the disease phenotype. The polymorphisms may be analyzed to determine if one or more is causing or contributing to the phenotype.

Microarray based technologies may be used at each step of the analysis. Mapping arrays which have a preselected set of known SNPs distributed approximately evenly across a genome may be used to identify a genomic region that is linked to a disease phenotype. That region may be analyzed by more targeted genotyping approaches. One approach that may be used is to resequence the area from a plurality of individuals. Novel polymorphisms in affected individuals may be identified during the resequencing. Methods of detecting polymorphisms are disclosed, for example, in U.S. Pat. Nos. 5,858,659, 5,925,525, and 6,300,063.

The American Diabetes Association has sponsored a multicenter project, Genetics of NIDDM (GENNID), to collect samples from diabetes patients and their relatives in four American populations. See, Raffel et al., Diabetes Care 19:864 872 (1996). We have carried out linkage studies with more than 10,000 SNP markers among 117 individuals from Hispanic/Latino diabetes families. Multipoint nonparametric linkage analysis was performed with MERLIN (Abcasis et al. Nat. Gen. 30, 97-101, 2002) for affection status of diabetes. A region on chromosome 2q24 centered at 162.346cM was linked to diabetes with LOD=3.05. Additional linkage regions on chromosomes 17 and 3 were also identified. A genomewide linkage study using 389-395 microsatellite markers for these populations (Gelder Ehm et al. Am. J. Hum. Genet. 66, 1871-1881, 2000) failed to identify significant linkage on chromosome 2 for this population. LOD is the log of the odds ratio of the probability of the data occurring under the specific hypothesis relative to the null hypothesis.

The gene at the peak of linkage was identified as formin homology 2 domain containing 2 (FHOD2 also called FMNL2). Formin homology (FH) or Diaphanous-related formin proteins are highly structured proteins and components of RHO family GTPase signaling pathways that affect cytoskeletal organization and induce transcriptional activation of the serum response element (SRE). Eukaryotic FH proteins consist of several conserved domains that are organized in a precise order. From the N terminus, FH proteins contain a loosely conserved FH3 domain, a GTPase binding domain, highly conserved FH1 and FH2 domains, a coiled-coil, and an autoregulatory domain. The C-terminal autoregulatory domain interacts with residues within and/or between the FH3 and the GTPase binding domain and thus mask the conserved FH1 and FH3 domains. This interaction was associated with kinase signaling pathways, actin-binding proteins and microtubules. Activation of the proteins by truncation of the C-terminal stimulated SRE-mediated transcriptional activation.

FHOD2 was predicted as KIAA1902 from cDNA clones (Nagase et al. DNA Res. 8, 179-87, 2001) (GenBank accession number XP_—057927) and recently identified and characterized in silico (Katoh and Katoh, Int J Oncol. 22, 1161-8, 2003). It is not well studied compared to another formin homologue protein, formin homolog overexpressed in spleen (FHOS). Besides spleen, FHOS is also highly expressed in skeletal muscle, lung and other tissues. FHOS was recently found to interact with the N-terminal cytoplasmic domain of insulin-responsive aminopeptidase (IRAP) with its C-terminal domain (Mol. Endocrinol. 17, 1216-29, 2003). It may mediate an interaction between glucose transporter isoform type 4 (GLUT4)/IRAP-containing vesicles and cytoskeleton and participate in exocytosis and/or retention of this compartment. Considering the highly conserved structures and domain sequences among FH proteins, FHOD2 may be similar to or share functionalities with FHOS and may play a key role in glucose homeostasis.

KIAA1902 was initially identified and sequences as a cDNA clone in a library generated from human brain. The predicted function based on homology search was in cell signaling and communication as a serine/threonine kinase receptor type 1. It was found to be highly expressed in all brain regions examined in the Nagase et al. study.

Three common variants among the 10K SNPs fell into the FHOD2 region, each with high LOD score, SNP_A-1511102 (TSC1457378, rs2345903) mapped to 5′UTR, SNP_A-1507899 (TSC0061688, rs727327) and SNP_A-1507852 (TSC0061687, rs727326) mapped to intron. The FMNL2 locus is found in the 314,364 base pairs of SEQ ID NO: 3 which is base pairs 3,401,401 to 3,715,764 of a chromosome 2 contig with GenBank accession number NT_—005403.

FHOD2 exons are from positions 1 to 252,186475 to 186558, 207271 to 207351, 213553 to 213629, 223272 to 223355, 225415 to 225567, 239668 to 239776, 243420 to 243496, 245488 to 245581, 271871 to 271945, 276027 to 276137, 279383 to 279532, 281623 to 281724, 283378 to 283690, 284041 to 284250, 289970 to 290094, 291057 to 291259, 292831 to 293065, 294188 to 294256, 294357 to 294437, 296540 to 296669, 300959 to 301122, 302105 to 302206, 304490 to 304588, 305323 to 305446, and 312328 to 314364.

The FHOD2 coding sequence is represented by positions 136 to 252,186475 to 186558, 207271 to 207351, 213553 to 213629, 223272 to 223355, 225415 to 225567, 239668 to 239776, 243420 to 243496, 245488 to 245581, 271871 to 271945, 276027 to 276137, 279383 to 279532, 281623 to 281724, 283378 to 283690, 284041 to 284250, 289970 to 290094, 291057 to 291259, 292831 to 293065, 294188 to 294256, 294357 to 294437,296540 to 296669, 300959 to 301122, 302105 to 302206, 304490 to 304588, 305323 to 305446, and 312328 to 312437.

Variants in the region of FHOD2 have been identified and documented in public databases. For example, dbSNP, lists 922 SNPs in the FMNL2 gene (contig positions 3401401 to 3715764) and 5 variants in the genomic region of FMNL2. Table 1 contains a reference identification number for each of the 927 SNPs in the region.

	TABLE 1
	A	B	C	D	E
1	rs2463	rs2043741	rs2678295	rs4254462	rs4664570
2	rs715046	rs2068886	rs2678296	rs4260194	rs4664573
3	rs715047	rs2078444	rs2678297	rs4274556	rs4664574
4	rs727326	rs2083098	rs2678298	rs4289130	rs4664575
5	rs727327	rs2083099	rs2678299	rs4293523	rs4664576
6	rs727601	rs2118376	rs2678300	rs4297832	rs4664577
7	rs727602	rs2164402	rs2678301	rs4311022	rs4664578
8	rs727603	rs2272535	rs2678302	rs4311023	rs4664579
9	rs734540	rs2272536	rs2678303	rs4328591	rs4664580
10	rs734541	rs2304556	rs2678304	rs4331457	rs4664581
11	rs746780	rs2304557	rs2678305	rs4335900	rs4664582
12	rs747012	rs2304558	rs2881328	rs4359594	rs4664583
13	rs751392	rs2345872	rs2881335	rs4368285	rs4664584
14	rs893328	rs2345873	rs2881336	rs4371310	rs4664585
15	rs893329	rs2345874	rs2881338	rs4371312	rs4664586
16	rs893330	rs2345875	rs2881339	rs4396660	rs4664587
17	rs893331	rs2345902	rs3047878	rs4425036	rs4664588
18	rs920244	rs2345903	rs3047882	rs4426480	rs4664589
19	rs934750	rs2346181	rs3047921	rs4442949	rs4664590
20	rs934751	rs2346182	rs3047928	rs4444472	rs4664591
21	rs1023754	rs2346183	rs3047933	rs4471845	rs4664592
22	rs1065266	rs2346184	rs3075786	rs4482430	rs4664593
23	rs1155778	rs2346185	rs3080597	rs4519464	rs4664594
24	rs1155779	rs2346186	rs3080598	rs4520990	rs4664595
25	rs1344152	rs2346198	rs3080600	rs4528717	rs4664596
26	rs1344153	rs2346199	rs3080632	rs4538152	rs4664597
27	rs1370497	rs2346200	rs3080633	rs4544376	rs4664599
28	rs1370499	rs2346201	rs3080636	rs4547483	rs4664600
29	rs1370500	rs2346202	rs3215380	rs4547484	rs4992311
30	rs1370501	rs2346203	rs3215381	rs4613221	rs5008216
31	rs1370502	rs2346204	rs3748941	rs4637059	rs5835420
32	rs1370503	rs2346205	rs3762622	rs4664100	rs5835421
33	rs1370504	rs2346206	rs3811568	rs4664101	rs5835422
34	rs1437679	rs2346207	rs3811569	rs4664102	rs5835423
35	rs1437680	rs2346208	rs3811570	rs4664103	rs5835424
36	rs1437681	rs2346209	rs3811571	rs4664104	rs5835425
37	rs1465818	rs2346532	rs3811572	rs4664105	rs5835426
38	rs1529998	rs2346533	rs3811573	rs4664106	rs5835427
39	rs1545134	rs2459776	rs3811574	rs4664107	rs5835428
40	rs1561267	rs2577175	rs3811575	rs4664108	rs5835429
41	rs1579050	rs2577176	rs3811576	rs4664109	rs5835430
42	rs1812713	rs2577177	rs3811577	rs4664110	rs5835431
43	rs1837105	rs2577178	rs3817620	rs4664111	rs5835432
44	rs1866115	rs2577179	rs3841082	rs4664112	rs5835433
45	rs1866116	rs2577180	rs3845678	rs4664113	rs5835434
46	rs1866117	rs2577181	rs3890370	rs4664114	rs5835435
47	rs1878632	rs2577182	rs3901192	rs4664115	rs5835436
48	rs1965785	rs2577183	rs4035882	rs4664116	rs5835437
49	rs1985975	rs2577184	rs4143274	rs4664117	rs5835438
50	rs2003332	rs2577185	rs4146259	rs4664118	rs5835439
51	rs2003333	rs2577187	rs4146903	rs4664120	rs5835440
52	rs2028168	rs2678294	rs4233659	rs4664569	rs5835441
53	rs6433983	rs6715576	rs6742066	rs7425826	rs7595822
54	rs6433984	rs6715843	rs6742846	rs7426043	rs7596254
55	rs6433985	rs6716773	rs6743018	rs7558989	rs7596408
56	rs6433986	rs6717982	rs6744391	rs7559217	rs7599894
57	rs6433990	rs6718081	rs6744977	rs7561292	rs7600114
58	rs6434003	rs6718337	rs6745613	rs7561791	rs7600425
59	rs6434017	rs6718584	rs6745714	rs7562904	rs7600568
60	rs6434025	rs6718773	rs6746190	rs7563675	rs7600624
61	rs6434028	rs6720219	rs6746284	rs7565316	rs7601047
62	rs6434042	rs6720532	rs6746499	rs7571665	rs7602505
63	rs6434046	rs6721601	rs6746538	rs7571693	rs7602756
64	rs6434059	rs6721741	rs6746777	rs7573833	rs7602975
65	rs6434060	rs6722740	rs6746800	rs7573890	rs7604573
66	rs6434061	rs6724305	rs6747312	rs7574348	rs7605220
67	rs6434062	rs6724909	rs6747765	rs7574699	rs7606289
68	rs6434068	rs6725060	rs6748954	rs7575177	rs7606397
69	rs6434078	rs6725656	rs6748966	rs7575441	rs7606592
70	rs6434081	rs6725661	rs6749146	rs7576442	rs7607120
71	rs6434082	rs6725789	rs6750956	rs7576568	rs7607429
72	rs6434097	rs6725954	rs6751094	rs7576847	rs7607506
73	rs6434098	rs6727037	rs6751151	rs7579097	rs7607747
74	rs6434099	rs6727781	rs6751576	rs7579243	rs7608137
75	rs6434113	rs6728118	rs6751778	rs7579511	rs7608463
76	rs6434114	rs6729565	rs6751924	rs7579527	rs7608585
77	rs6434115	rs6729611	rs6754501	rs7580023	rs7609122
78	rs6434128	rs6729957	rs6754723	rs7581544	rs9288090
79	rs6434129	rs6730004	rs6755173	rs7581918	rs9288103
80	rs6704965	rs6730982	rs6756108	rs7582423	rs9288107
81	rs6705427	rs6731407	rs6756332	rs7582905	rs9288108
82	rs6705986	rs6731516	rs6756367	rs7582984	rs9677373
83	rs6706132	rs6731699	rs6756465	rs7583872	rs9677380
84	rs6706231	rs6731738	rs6757207	rs7584000	rs9678076
85	rs6706394	rs6732842	rs6757219	rs7585911	rs9678127
86	rs6706407	rs6733002	rs6757782	rs7586195	rs9679683
87	rs6706416	rs6733442	rs6757784	rs7586834	rs9752336
88	rs6707033	rs6734194	rs6757877	rs7587450	rs9753431
89	rs6707730	rs6734434	rs6757879	rs7587658	rs9967683
90	rs6708443	rs6734534	rs6757884	rs7587794	rs9967814
91	rs6708667	rs6736003	rs6759772	rs7588008	rs9967819
92	rs6709494	rs6736639	rs6759858	rs7589218	rs9989874
93	rs6710417	rs6736856	rs6760139	rs7590079	rs10048775
94	rs6710651	rs6737006	rs6760242	rs7590207	rs10165830
95	rs6710689	rs6737699	rs6760321	rs7591466	rs10166430
96	rs6710999	rs6738344	rs6760818	rs7591606	rs10166703
97	rs6712716	rs6739300	rs6761178	rs7591723	rs10168692
98	rs6712717	rs6739525	rs6761708	rs7591868	rs10168793
99	rs6713348	rs6739557	rs6761815	rs7592127	rs10169491
100	rs6713711	rs6739954	rs7368679	rs7592283	rs10169514
101	rs6713836	rs6740909	rs7371728	rs7593411	rs10171204
102	rs6714213	rs6741131	rs7421103	rs7593505	rs10172230
103	rs6714218	rs6741664	rs7421149	rs7593580	rs10173398
104	rs6715038	rs6741728	rs7424540	rs7594969	rs10173899
105	rs10174137	rs10497111	rs11326775	rs11883839	rs12474369
106	rs10177081	rs10524595	rs11342086	rs11884490	rs12474927
107	rs10178618	rs10538950	rs11343380	rs11885624	rs12474974
108	rs10179385	rs10539703	rs11344436	rs11886407	rs12476186
109	rs10179391	rs10539716	rs11345920	rs11891914	rs12476261
110	rs10179418	rs10554817	rs11349892	rs11892222	rs12478426
111	rs10180234	rs10559962	rs11360283	rs11892232	rs12478681
112	rs10180542	rs10567754	rs11369015	rs11892322	rs12612608
113	rs10182254	rs10580348	rs11384938	rs11892659	rs12613219
114	rs10182858	rs10581091	rs11389713	rs11892662	rs12613900
115	rs10182993	rs10581805	rs11390817	rs11892859	rs12613901
116	rs10183530	rs10584642	rs11392993	rs11892925	rs12613902
117	rs10184210	rs10597937	rs11403468	rs11893683	rs12614414
118	rs10184459	rs10600592	rs11407117	rs11897348	rs12614465
119	rs10185455	rs10605671	rs11408358	rs11897616	rs12614608
120	rs10186650	rs10605918	rs11408981	rs11897929	rs12616141
121	rs10188611	rs10617862	rs11428785	rs11898429	rs12616382
122	rs10191051	rs10624832	rs11428942	rs11898643	rs12616778
123	rs10191361	rs10630545	rs11430178	rs11899045	rs12616906
124	rs10193104	rs10645049	rs11436361	rs11899068	rs12617686
125	rs10193625	rs10646636	rs11438121	rs11899286	rs12618643
126	rs10194603	rs10649381	rs11440524	rs11901239	rs12618892
127	rs10195380	rs10670761	rs11446146	rs11901392	rs12619061
128	rs10195727	rs10672047	rs11452085	rs11901762	rs12619679
129	rs10195938	rs10685899	rs11455357	rs11902693	rs12619706
130	rs10200831	rs10688923	rs11462086	rs11904088	rs12621431
131	rs10203197	rs10694393	rs11538551	rs11904617	rs12621715
132	rs10204038	rs10699311	rs11674872	rs12052225	rs12622247
133	rs10206228	rs10702331	rs11675841	rs12052587	rs12622731
134	rs10207275	rs10717513	rs11676208	rs12052694	rs12623070
135	rs10208744	rs10717805	rs11676897	rs12052809	rs12623225
136	rs10208776	rs10719527	rs11678890	rs12104452	rs12693359
137	rs10209069	rs10803964	rs11679491	rs12105467	rs12693362
138	rs10209763	rs10931054	rs11680639	rs12105483	rs12693378
139	rs10210776	rs10931062	rs11682487	rs12151546	rs12693393
140	rs10211164	rs10931067	rs11684450	rs12151547	rs12693404
141	rs10432492	rs10931068	rs11684983	rs12151851	rs12693405
142	rs10432493	rs10931069	rs11686482	rs12185697	rs12693406
143	rs10432494	rs10931075	rs11687067	rs12464681	rs12693407
144	rs10439306	rs10931079	rs11687886	rs12465320	rs12693415
145	rs10460316	rs10931089	rs11688690	rs12466536	rs12693429
146	rs10497100	rs10931146	rs11689419	rs12467610	rs12986786
147	rs10497101	rs10931150	rs11692343	rs12467702	rs12987041
148	rs10497102	rs10931154	rs11692786	rs12468231	rs12987348
149	rs10497103	rs10931163	rs11694392	rs12468606	rs12987493
150	rs10497104	rs10931189	rs11694512	rs12468749	rs12987733
151	rs10497105	rs10931191	rs11694575	rs12468789	rs12987825
152	rs10497106	rs11267395	rs11694760	rs12471183	rs12988475
153	rs10497107	rs11275578	rs11695776	rs12471256	rs12989510
154	rs10497108	rs11292635	rs11696004	rs12471268	rs12989726
155	rs10497109	rs11306212	rs11883762	rs12471699	rs12989840
156	rs10497110	rs11325472	rs11883821	rs12471988	rs12989943
157	rs12990922	rs13023975	rs13408985	rs13021931	rs13403239
158	rs12990935	rs13024308	rs13409788	rs13022419	rs13404703
159	rs12991039	rs13024504	rs13409795	rs13022422	rs13404820
160	rs12991203	rs13024533	rs13409883	rs13023506	rs13405110
161	rs12991474	rs13026023	rs13409884	rs13023529	rs13405341
162	rs12992255	rs13026560	rs13409889	rs13023659	rs13405868
163	rs12992391	rs13026712	rs13409892	rs13023779	rs13406875
164	rs12992900	rs13026858	rs13412508	rs13023928	rs13406882
165	rs12993239	rs13027160	rs13412574	rs13023943	rs13406999
166	rs12993871	rs13027356	rs13413144
167	rs12994519	rs13028185	rs13414252
168	rs12995460	rs13028472	rs13414680
169	rs12998222	rs13028480	rs13414887
170	rs12999189	rs13028846	rs13415463
171	rs12999503	rs13029234	rs13415525
172	rs13000076	rs13029407	rs13417781
173	rs13000092	rs13029667	rs13418521
174	rs13000852	rs13029771	rs13418748
175	rs13001913	rs13031546	rs13418821
176	rs13002771	rs13033172	rs13418832
177	rs13003413	rs13033209	rs13418936
178	rs13003885	rs13033468	rs13419000
179	rs13004473	rs13034458	rs13419948
180	rs13004494	rs13034520	rs13421428
181	rs13005838	rs13034665	rs13423608
182	rs13006032	rs13382410	rs13423731
183	rs13006671	rs13386403	rs13424038
184	rs13007956	rs13387295	rs13425532
185	rs13009223	rs13387298	rs13425748
186	rs13009580	rs13387427	rs13426403
187	rs13010632	rs13387542	rs13426471
188	rs13011152	rs13388095	rs13427076
189	rs13011684	rs13390698	rs13427289
190	rs13011850	rs13391023	rs13427485
191	rs13014071	rs13396439	rs13427489
192	rs13015675	rs13396834	rs13427942
193	rs13016534	rs13397100	rs13427990
194	rs13017355	rs13397890	rs13428569
195	rs13017492	rs13398440	rs13429157
196	rs13018337	rs13400908	rs13430331
197	rs13018434	rs13401202	rs13430448
198	rs13018870	rs13402243	rs13430837
199	rs13020285	rs13403205	rs13432002

Each of these SNPs is either in an intronic region of an untranslated region. Methods to identify polymorphisms in the genomic region containing FHOD2 that are associated with a risk for type 2 diabetes or IH are disclosed. Polymorphisms that are associated with a decreased risk of type 2 diabetes may also be identified. Methods of identifying polymorphisms are well known in the art. In one embodiment the genomic region is resequenced in a plurality of individuals.

This work represents the first to associate FHOD2 with common type 2 diabetes. The results indicate that FHOD2 may be involved in the transcription control of glucose response elements and in some embodiments FHOD2 is used as a target for drugs to treat diabetes and insulin resistance. In some embodiments variations in or around this gene may be associated with the severity and progression of impaired glucose homeostasis. In some embodiments variations in or near this gene may be used to diagnosis diabetes or insulin resistance. In some embodiments variations in or near this gene may be used to predict patient outcome or to predict treatment outcome. In some embodiments variations in or near this gene may be used in one or more pharmacogenetic tests.

FHOD2 may be used in methods to identify compounds useful in the treatment of diabetes and related diseases, methods to determine the predisposition of individuals to diabetes and methods for diagnosis and prognosis of diabetes.

In some embodiments one or more probes to the FHOD2 gene are disclosed. The probes may be for example, oligonucleotides that may be, for example, 20, 25, 30, 50, 60 or 100 bases. The probes are complementary to a region of the FHOD2 gene. The probes may be complementary to either strand of the double stranded DNA. The probes may be complementary to one or more forms of the FHOD2 mRNA. The probes may be complementary to an intron of the FHOD2 gene or to the 5′UTR or 3′UTR of FHOD2. In a preferred embodiment one or more probes that are complementary to a polymorphic form of FHOD2 that is associated with a metabolic disease are disclosed. Probe pairs that include a probe to a wild type form and a probe to a mutant form may be used.

In one embodiment a SNP that is indicative of a haplotype that is associated with a metabolic disorder, for example with type 2 diabetes are disclosed. Haplotypes are described in Gabriel et al., Science 296: 2225-9 (2002), Daly et al. Nat Genet. 29: 229-32 (2001) and Rioux et al. Nat Genet. 29:223-8 (2001), which are each incorporated herein in their entireties by reference for all purposes.

In one embodiment probes that are complementary to the FHOD2 gene in a polymorphic region are disclosed. The probes may be used to genotype polymorphisms in or near FHOD2. Allele specific probes may be used so that one probe hybridizes specifically to one allele of a biallelic polymorphism and another probe hybridizes specifically to a second allele of the biallelic polymorphism. In one embodiment, a plurality of probes wherein at least one probe is complementary to one allele of a biallelic polymorphism in the FHOD2 gene or the region surrounding the FHOD2 gene, within 1000, 2000, 3000 or 5000 bases of the FHOD2 gene, and at least one probe is complementary to a second allele is disclosed. The presence or absence of one or more allele may be detected. The presence of a specific allele of a polymorphism in the region may be associated with diabetes.

In one embodiment a kit for genotyping at least one polymorphism in FHOD2 is disclosed. The kit may comprise a probe that hybridizes to a polymorphic region of the FHOD2 gene, including as non limiting examples, the coding region, the 5′UTR, the 3′UTR, introns and upstream or downstream regulatory regions. The kit may comprise a probe that hybridizes to the FHOD2 gene immediately adjacent to a polymorphism. The probe may further comprise a tag sequence.

Methods of genotyping a polymorphism are known to those of skill in the art and any method of genotyping may be used to determine which alleles are present at one or more polymorphic positions in and around the FHOD2 gene. Genotyping methods may involve methods such as oligo ligation assay (OLA), single base extension (SBE), allele specific hybridization, sequencing, and mass spectroscopy. For additional methods of genotyping see Syvanen, A-C, Nat. Rev. Genet. 2:930-942 (2001), Jenkins and Gibson, Comp. Funct. Genom. 3:57-66 (2002), and Twyman and Primrose, Pharmacogenomics, 4:67-79 (2003), each of which is incorporated herein by reference in its entirety for all purposes. Kits comprising oligonucleotides for the genotyping of polymorphisms in the FHOD2 gene and surrounding region are contemplated herein.

In some embodiments the kit comprises an array. The array may comprise probes for genotyping one or more polymorphisms in the FHOD2 gene and surrounding DNA. The array may also comprise probes for genotyping other polymorphisms in other genes that are predicted to be susceptibility genes for diabetes or for other metabolic disorders. In one embodiment an assay comprising oligonucleotides for a plurality of polymorphisms that are indicative of susceptibility for diabetes is disclosed.

For a discussion of genotyping analysis methods see, for example, Elena and Lenski Nature Reviews, Genetics 4:457-469 (2003), Hirschorn et al. Genetics in Medicine 4:45-61 (2002) and Glazier et al. Science 298:2345-2349 (2002) each of which is incorporated herein by references for all purposes. One method of allele specific genotyping that may be used to genotype selected SNPs in the FHOD2 region is described in Hardenbol et al. Nat Biotechnol. 2003 Jun; 21(6):673-8. Epub May 5, 2003 and in U.S. Patent Publication No. 20040101835. The molecular inversion probe (MIP) assay described in Hardenbol et al. may be used to genotype large numbers of SNPs. Multiplex analysis of more than 1,000, 5,000 or 10,000 probes in a single tube may be performed using the assay. A single probe is used per marker or SNP. The genotypes may be read out using a tag array such as the Affymetrix GenFlex or Tag 3 array. Molecular inversion probes may be designed for a selected subset of SNPs, for example MIPs can be designed and synthesized for more than 50, 100, or 500 of the SNPs in Table 1. Novel SNPs identified by resequencing may be targeted by MIPs and the MIP assay. Smaller numbers of SNPs in the FHOD2 region may be genotyped by any method known in the art.

Polymorphisms in the FHOD2 gene may be used to stratifyhuman patients according to risk of developing type 2 diabetes or metabolic disorders, as such methods relate to indicators of risk for diabetes associated with the FHOD2 gene and polymorphic variants in the FHOD2 gene and in the surrounding region are also disclosed.

Conclusion

The present inventions provide isolated nucleic acid sequences, probes and methods for identifying mutations that are associated with an increased risk of type 2 diabetes. It is to be understood that the above description is intended to be illustrative and not restrictive. Many variations of the invention will be apparent to those of skill in the art upon reviewing the above description. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. An isolated nucleic acid comprising a polymorphic variant of SEQ ID NO: 2 wherein the polymorphic variant is associated with a metabolic disorder.

2. The isolated nucleic acid of claim 1 wherein said polymorphic variant is a single nucleotide polymorphism.

3. The isolated nucleic acid sequence of claim 1 wherein the metabolic disorder is altered glucose homeostasis.

4. The isolated nucleic acid sequence of claim 1 wherein the metabolic disorder is type 2 diabetes.

5. A protein encoded by the isolated nucleic acid of claim 1.

6. The protein of claim 5 wherein the polymorphic variant is associated with increased risk of a type 2 diabetes.

7. The protein of claim 5 wherein the polymorphic variant is associated with increased altered glucose homeostasis.

8. An antibody to the protein of claim 5 wherein the protein varies at one amino acid.

9. An antibody to the protein of claim 5 wherein the protein is a truncated form of the protein or wherein the protein has a deletion of up to 10, 20, 30, 50 or 100 amino acids.

10. An antibody to the protein of claim 5 wherein the protein varies at two or more amino acids.

11. An oligonucleotide probe comprising 20 to 100 contiguous nucleotides of a polymorphic variant of SEQ ID NO. 3 or its complement, wherein the probe is complementary to a region comprising the polymorphism and is complementary to a polymorphic variant that is associated with a metabolic disorder.

12. The oligonucleotide probe of claim 11 wherein the metabolic disorder is type 2 diabetes.

13. The oligonucleotide probe of claim 12 wherein the probe is 20 to 50 nucleotides in length.

14. An oligonucleotide probe comprising 20 to 100 contiguous nucleotides of a polymorphic variant of SEQ ID NO. 2 or its complement, wherein the probe is complementary to a region comprising the polymorphism and is complementary to a polymorphic variant that is associated with a metabolic disorder.

15. The oligonucleotide probe of claim 14 wherein the metabolic disorder is type 2 diabetes.

16. The oligonucleotide probe of claim 14 wherein the probe is 20 to 50 nucleotides in length.

17. The oligonucleotide probe of claim 11 wherein the polymorphism is a haplotype tag SNP that is indicative of the presence of a haplotype that is associated with type 2 diabetes.

18. A method of determining if a patient is at increase risk of developing type 2 diabetes comprising:

identifying a risk allele of a polymorphic variant of SEQ ID NO 2 or SEQ ID NO 3 that is associated with an increased risk of developing type 2 diabetes;

determining if the risk allele is present in the patient; and

determining that the patient is at increase risk of developing type 2 diabetes if the risk allele is present.

19. The method of claim 18 wherein said identifying step comprises:

resequencing at least 100,000 bases of SEQ ID NO 3 in a plurality of individuals that have type 2 diabetes;

comparing the sequences obtained to a reference sequence from a healthy individual; and

identifying at least one sequence variant that is present in at least one individual that has type 2 diabetes and absent in the reference sequence, wherein the sequence variant is indicative of a risk allele.

20. The method of claim 19 wherein at least 300,000 bases of SEQ ID NO 3 is resequenced.